Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the intramolecular cyclization of N-terminal glutamine residues into pyroglutamic acid (pGlu*) liberating ammonia. A QC was first isolated by Messer from the latex of the tropical plant Carica papaya in 1963 (Messer, M. 1963 Nature 4874, 1299). 24 years later, a corresponding enzymatic activity was discovered in animal pituitary (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). For the mammalian QC, the conversion of Gln into pGlu by QC could be shown for the precursors of TRH and GnRH (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536; Fischer, W. H. and Spiess, J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). In addition, initial localization experiments of QC revealed a co-localization with its putative products of catalysis in bovine pituitary, further improving the suggested function in peptide hormone synthesis (Bockers, T. M. et al. 1995 J Neuroendocrinol 7, 445-453). In contrast, the physiological function of the plant QC is less clear. In the case of the enzyme from C. papaya, a role in the plant defense against pathogenic microorganisms was suggested (El Moussaoui, A. et al. 2001 Cell Mol Life Sci 58, 556-570). Putative QCs from other plants were identified by sequence comparisons recently (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36). The physiological function of these enzymes, however, is still ambiguous.
The QCs known from plants and animals show a strict specificity for L-Glutamine in the N-terminal position of the substrates and their kinetic behavior was found to obey the Michaelis-Menten equation (Pohl, T. et al. 1991 Proc Natl Acad Sci USA 88, 10059-10063; Consalvo, A. P. et al. 1988 Anal Biochem 175, 131-138; Gololobov, M. Y. et al. 1996 Biol Chem Hoppe Seyler 377, 395-398). A comparison of the primary structures of the QCs from C. papaya and that of the highly conserved QC from mammals, however, did not reveal any sequence homology (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36). Whereas the plant QCs appear to belong to a new enzyme family (Dahl, S. W. et al. 2000 Protein Expr Purif 20, 27-36), the mammalian QCs were found to have a pronounced sequence homology to bacterial aminopeptidases (Bateman, R. C. et al. 2001 Biochemistry 40, 11246-11250), leading to the conclusion that the QCs from plants and animals have different evolutionary origins.
Recently, it was shown that recombinant human QC as well as QC-activity from brain extracts catalyze both, the N-terminal glutaminyl as well as glutamate cyclization. Most striking is the finding, that cyclase-catalyzed Glu1-conversion is favored around pH 6.0 while Gln1-conversion to pGlu-derivatives occurs with a pH-optimum of around 8.0. Since the formation of pGlu-AR-related peptides can be suppressed by inhibition of recombinant human QC and QC-activity from pig pituitary extracts, the enzyme QC is a target in drug development for treatment of Alzheimer's disease.
U.S. Pat. No. 7,572,614 (Wang et al) and Huang et al (2005) PNAS 102(37), 13117-13122 both describe one example of the crystal structure of soluble glutaminyl cyclase. The crystal structure disclosed in Wang et al and Huang et al was generated using a protein expressed in E. coli, which results in a lack of glycosylation. It is well known that all mammalian QC contain at least one glycosylation site (Pohl, T. et al. (1991) Proc Natl Acad Sci USA 88, 10059-10063; Song, I. et al. (1994) J Mol Endocrinol 13, 77-86), which is glycosylated in the QC crystallized according to the present disclosure by virtue of being expressed in eukaryotic hosts, which can be observed in the crystal structures presented herein. In addition, all mammalian QCs contain two conserved cysteine residues close to the active site, which form a disulfide bond. In the crystal structure of Wang et al and Huang et al, the disulfide bond is lacking. The expression of mammalian secretory proteins in bacteria frequently results in the absence of disulfide formation (Hannig, G. and Makrides, S. C. (1998) Trends Biotechnol 16, 54-60). The disulfide bond is clearly present in both the human and murine QC crystal structures presented herein. Notably, the mutational analyses provided in the examples described herein suggest an important stabilizing function of the disulfide bond upon the overall structure. Furthermore, in the structure of Wang et al and Huang et al, a segment of residues (L205-H206-W207) close to the active site appears in two different conformations. Due to the orientations, the binding mode of substrates is affected and reliable mechanistic conclusions could not be drawn (Huang et al., 2005). The residue W207 is conserved in mammalian QCs (W208 in murine QC). In the human and murine QC crystal structures presented herein, the orientation is identical, although the adjacent residues L205 and H206 are not conserved. Therefore, the structural orientation of residues appears non-native in the crystal structure of Wang et al and Huang et al.
The expression of the murine and human QC in an eukaryotic host, as described in the present disclosure, allows the crystallization and structural refinement of a native mammalian QC and, importantly, unambiguous determination of the binding modes of inhibitors, as exemplified by the structural resolution of murine QC with three different inhibitory compounds (listed in Table 1 as Inhibitor A, Inhibitor B and Inhibitor C), which have similar inhibitory potency between human and murine QC.
In contrast to the structures described in previous publications by Wang et al and Huang et al., the present disclosure shows that the post-translational modifications disulfide formation and glycosylation lead to a single structural arrangement of the residues L205-H206-W207 in human and murine QC. The residues have a direct effect on the binding mode of active-site directed compounds. The multiple orientations of W207 in previous studies, led to variances in the binding modes of active-site-directed compounds.
TABLE 1Ki-values [μM] of selected inhibitorsfor murine (mQC) and human QC (hQC)InhibitormQChQCInhibitor A0.173 0.0542Inhibitor Bn.d.0.0613Inhibitor C0.05130.106
In contrast, unambigous binding modes of compounds and orientations of W207 were observed with three different inhibitors and even in a structure without inhibitor bound. Thus, the methods as described in the present disclosures provide substantial advances for structure-driven drug design of QC inhibitors.
The conclusion is particularly strengthened by the structural assessment of QCs from two different mammals.